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Nat Methods. 2006 Dec;3(12):1001-6. Epub 2006 Nov 5.

A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer.

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Nuffield Department of Clinical Medicine and Medical Research Council, Human Immunology Unit, Weatherall Institute of Molecular Medicine, The University of Oxford, Oxford Radcliffe Hospital, Oxford, OX3 9DU, UK.


Bioluminescence resonance energy transfer (BRET), which relies on nonradiative energy transfer between luciferase-coupled donors and GFP-coupled acceptors, is emerging as a useful tool for analyzing the quaternary structures of cell-surface molecules. Conventional BRET analyses are generally done at maximal expression levels and single acceptor/donor ratios. We show that under these conditions substantial energy transfer arises from random interactions within the membrane. The dependence of BRET efficiency on acceptor/donor ratio at fixed surface density, or expression level at a defined acceptor/donor ratio, can nevertheless be used to correctly distinguish between well-characterized monomeric and oligomeric proteins, including a very weak dimer. The pitfalls associated with the nonrigorous treatment of BRET data are illustrated for the case of G protein-coupled receptors (GPCRs) proposed to form homophilic and/or mixed oligomers on the basis of previous, conventional BRET experiments.

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